Figure 5.

Double immunostaining of lens sections with two rabbit primary antibodies (A, C) and controls in which the second primary antibody was omitted (B, D). A. Rab5B (red), pSmad1 (green). B. Rab5B (red), no pSmad1 primary antibody. No green staining from the second anti-rabbit secondary antibody was detected. C. Rab7 (green), Rab5B (red), TOTO-1 (blue). D. Rab7 (green), no Rab5B primary antibody, TOTO-1 (blue). No red staining from the second anti-rabbit secondary antibody was detected. E. Diagram illustrating the method used for double labeling (a) and the alternative outcomes of the control studies for specificity (b, c). In a, the first rabbit primary antibody (light blue, 1) binds to its antigen and is localized by a fluorescent-labeled anti-rabbit secondary antibody (red, 2). The tissue is washed, fixed in formalin and washed again. The second rabbit primary antibody is then added (yellow), which binds to the second antigen (3). The second anti-rabbit secondary antibody (green) is added and binds to the second rabbit primary antibody (4). The sequence shown in b and c illustrates the controls used in the present study. The first two steps are as in a, but the second rabbit primary antibody is omitted (3), providing no antibody for the second anti-rabbit fluorescent antibody to bind (green, 4). In b, the second fluorescent-labeled anti-rabbit secondary antibody (green) does not bind the first primary antibody (blue) and the section remains singly labeled. In the steps shown in c, the second fluorescent-labeled anti-rabbit secondary antibody (green) binds to the first rabbit primary antibody (blue), resulting in spurious "co-localization." Since staining by the second anti-rabbit secondary antibody was not seen, as shown in B and D, b accurately reflects the results obtained in the present studies; the events shown in c were not observed.